Serum-based, diagnostic, biological assay to predict pregnancy disorders

ABSTRACT

The invention provides serum-based, diagnostic, biological assays for predicting disorders of pregnancy resulting from poor trophoblast and/or placental ischemia, including preeclampsia. Serum samples from such subjects exhibit an ability to disrupt the architecture involving fetal trophoblasts and maternal endothelial cells in a three-dimensional, dual cell co-culture system provided herein, in contrast to normal pregnancy serum samples. Based on these distinctions, the assays are employed to predict pregnancy outcomes as early as first trimester.

This application is a continuation of U.S. application Ser. No.12/865,239, which is the National Stage of International Application No.PCT/US2009/000708, filed Feb. 4, 2009, which claims priority to U.S.Provisional Application No. 61/063,491 filed Feb. 4, 2008, thedisclosures of which are incorporated herein by reference in theirentirety.

BACKGROUND

Disorders of pregnancy pose a major public health problem because theyinvolve approximately 10% of human pregnancies. Preeclampsia (PE) is onesuch disorder which presents itself with maternal symptoms of globalendothelial disease, including glomeruloendotheliosis, liver andcerebral vascularitis. Diagnostic symptoms are high blood pressure(>140), proteinuria (>0.3 gm/ml) and general edema. (AGOG Committee onObstetric Practice, 2002) PE is a pregnancy-specific systemic disordercharacterized by a cascade of events and symptoms, including impairedtrophoblast invasion, decreased placental perfusion, placental ischemia,oxidative stress and imbalance in angiogenic and prothrombotic factorswhich can lead to apoptosis of trophoblasts⁽²⁰⁻²³⁾. Studies have alsoshown that in preeclampsia, there are elevated levels of circulating orplacental TNFα, IL-6, IL-8. IFNγ, leptin, perturbed renin angiotensinsystem, complement split products, antibodies to phospholipids, sFlt-1,soluble endoglin, IL12, decreased IL10, NO, hypoxia^((18, 24-31))amongst host of other factors. Uteroplacental abnormalities can resultin shallow placentation, poor spiral artery remodeling, and placentalischemia. PE is strictly a placental condition; it resolves afterdelivery. PE is diagnosed in the later half of pregnancy and therelatively late onset of clinical signs and a complex pathobiology of PEpresent obstacles for its study. There exist no concrete in vitro oranimal models that mirror the morphological and mechanisticunderpinnings of PE.

Gestational diabetes, intrauterine growth restriction, and placentalabruption are other pregnancy disorders associated with placentalangiogenic anomalies and ischemia. Gestational diabetes is characterizedby high blood glucose levels during pregnancy. About 3%-5% of allpregnant women in the U.S. are diagnosed with the condition, which isbelieved to result from hormonal changes that occur during pregnancy.Increases in hormone levels made in the placenta cause insulinresistance, which increases as the placenta grows larger and producesmore hormones. If the pancreas cannot produce enough insulin to overcomethe effect of the increased hormones during pregnancy, sugar levels willrise, resulting in gestational diabetes.

Placental abruption affects about 9 in 1,000 pregnancies. It can occurany time after the 20th week and results from a cascade ofpathophysiologic processes ultimately leading to the separation of theplacenta prior to delivery. Pregnancies complicated by abruption resultin increased frequency of low birth weight, preterm delivery,stillbirth, and perinatal death.⁽³²⁾ The causes are not well-understood;some women develop it without any identifiable cause. Known risk factorshigh blood pressure (140/90 mm Hg or higher), either chronic or causedby the pregnancy (either by pregnancy-induced hypertension orpreeclampsia).

Intrauterine growth retardation (IUGR), defined as less than 10 percentof predicted fetal weight for gestational age, may result in significantfetal morbidity and mortality if not properly diagnosed. The conditionis most commonly caused by inadequate maternal-fetal circulation, with aresultant decrease in fetal growth. Maternal causes of IUGR account formost uteroplacental cases. Chronic hypertension is the most common.Moreover, the infants of hypertensive mothers have a three-fold increasein perinatal mortality compared with infants with IUGR who are born ofnormotensive mothers. IURR also result from preeclampsia, which causesplacental damage that result in uteroplacental insufficiency due toluminal narrowing and medial degeneration, leading to diminished bloodflow to the developing infant. Consequently, the infants fail to grownormally. Treatment of the mother and the growth-restricted fetus istypically dictated by the etiology of the condition. Maternalhyperoxygenation has been evaluated in several studies and low-doseaspirin (150 mg per day) has also been studied.

Efforts have been made to provide assays for the diagnosis of PE.Numerous assays employ identification and/or measurement of variousbiochemical markers such as specific protein or nucleic acids inmaternal samples. Exemplary are U.S. Pat. Nos. 6,735,529; 6,620,590;6,495,330; and 6,258,540 and United States Patent Publication Nos.2007/0185200; 2004/0038305; 2007/01785302007/0104707; 2007/0020766; and2006/0183175. None appear to work with any consistent, reliable, degreeof success. Another known assay involves culturing human trophoblasts inthe presence or absence of a pregnant woman's serum or plasma andcomparing viability of the cells cultured. See United States PatentPublication No. 2005/0074746. Like the biochemical marker assays, thiscell-based assay is reported to be an inconsistent and unreliablepredictive measure for PE.

Efforts also have been made to provide mouse strains exhibit phenotypesassociated with various adverse pregnancy outcomes, including PE.Hayakawa et al. demonstrated high fetal resorption rates, hypertension,proteinuria and glomerular nephritis in pregnant BALB/C mice exposed toIL-12 stimulated splenocytes⁽¹⁴⁾. Takimoto et al. mated transgenic miceexpressing components of the human renin-angiotensin system, resultingin the development of PE manifestations⁽¹⁵⁾. Similarly, mice deficientin the cyclin-dependent kinase inhibitor p57kip1 exhibit some of thefeatures associated with PE⁽¹⁶⁾. Proteinuria, hypertension,glomerulosclerosis and small liter size were also noted in spontaneouslyhypertensive (BPH/5) matings⁽¹⁷⁾. However, these models do not addressthe issue of intrinsic response in wild type animals to circulatinginflammatory components and placenta-derived factors.

SUMMARY OF THE INVENTION

The invention provides a simple in vitro dual cell culture model thatmimics the invasion of fetal trophoblasts over endothelial cells inresponse to normal pregnancy serum. The invention is based on theunexpected observation that invasive extravillous trophoblasts (EVT)from first trimester spontaneously interact with maternal endothelialcells, allowing for trophoblastic “fingerprinting” of the endothelialcell architecture, which exhibits a characteristic vacuolization, whenthe two cell types are co-cultured on a soluble matrix with normalpregnancy serum. In contrast, third trimester and term trophoblasts donot exhibit this vacuolization in endothelial cell co-cultures. In theabsence of endothelial cells, trophoblasts themselves do not form sucharchitecture and remain as aggregates of cells in response to growthfactors from serum. Surprisingly, serum from pregnant female humans thatgo on to acquire complications like preeclampsia disrupts thecharacteristic vacuolization of the cells co-cultured. Thus, theinvention is based on the premise that maternal serum is a “blueprint”that mirrors the “global welfare” of placenta and fetus. Due totrans-placental transportation, any inflammatory milieu associated withplacental dysfunction would be released into circulation.

Although the invention is exemplified using preeclampsia (PE), for thereasons discussed above, the model also finds applicability in theprediction of any disorder stemming from either poor trophoblastinvasion or placental ischemia, or both, including for example PE,gestational diabetes, intrauterine growth restriction (IUGR) andplacental abruption. Other trophoblasitic diseases with hyper- or hypoinvasive features that are similar to the foregoing can also benefitfrom the methods and compositions of the invention.

Accordingly, in one aspect, the invention provides an assay forassessing whether a pregnant female is at risk of developing a disorderof pregnancy associated with trophoblast invasion or placental ischemia.Exemplary disorders include preeclampsia, gestational diabetes,intrauterine growth restriction and placental abruption. The assayincludes incubating a co-culture of human endothelial cells and humantrophoblast cells in the presence of serum or plasma obtained from apregnant female for a period of time sufficient to permit vacuolization(also referred to as “capillary formation” and “tube formation” in FIG.5), and after incubation determining whether substantial vacuolizationin the co-culture has occurred. By “substantial vacuolization” we meanhigh number of vacuoles. In this context, each vacuole is the smallcavity completely bound by elongated cellular structure as indicated inFIGS. 1 c and 5. As observed under microscope, vacuolization comprise ofthin walled vacuoles having few branch points. The branch points are thepoints from which multiple vacuoles are initiated/connected. Thequantity of tubes formed (also means number of vacuoles) will besubstantially less by comparison with normal pregnancy serum. Normalpregnancy serum will exhibit tube-vacuole formation over about 40tubes/well of a 48 well plate (for example between about 45 and 75tubes/well as can be seen in FIGS. 7 c and 11 a). This average number ofsuch tubes-vacuoles in response to normal pregnancy serum is defined as“normal endothelial-trophoblast cross-talk”. In contrast, serum orplasma from a pregnant female at risk for or having preeclampsia willexhibit tube-vacuole formation substantially less than about 40 tubesper well (for example between about 5 and 35 tubes/well as can be seenin FIGS. 7 c and 11 a). This average number of such tubes-vacuoles isdefined as “abnormal endothelial-trophoblast crosstalk”.

Optionally, an addition co-culture in the absence of serum from apregnant female can be performed under the same conditions and the twoco-cultures compared to determine whether vacuolization in the firstco-culture has occurred. Typical incubation time is between 10 and 12hours, in carbon dioxide at 37° C. and preferably the serum, in anamount ranging from 0.5 to ml will have been taken from the firsttrimester of the pregnant female's pregnancy. The assay can be performedon a natural or synthetic soluble matrix. Exemplary matrices includematrigel, collagen, fibronectin, elastin and combinations thereof.

The human endothelial cells employed may be from umbilical vein or theymay be uterine, myometrial, cedicual, aorta, microvascular, dermal orother endothelial cells that express VE-cadherin, PECAM, and/orAquaporin 1. The trophoblasts employed may be from primary villous orextravillous trophoblasts, HTR8, 3A or JEG3 trophoblasts or otherinvasive trophoblasts isolated from choriocarcinomas.

This simple assay, based on “cross-talk” between fetal and maternalcells at the fetal-maternal interface can be used as diagnostic tool topredict pregnancy outcomes as early as 7-10 weeks of pregnancy andimpending pregnancy complications like preeclampsia, intrauterine growthrestriction, gestational diabetes and placental abruption. The method issimple, non-invasive, cost effective, and can be completed within about8-12 hrs. It requires a one time draw of blood (1-5 ml) at differentstages of pregnancy, preferably between 7-21 weeks, so as to yield serumof about 0.3-5 ml, preferably about 0.5-2 ml for the experiment. Themethod does not require any major equipment and can be carried out byany diagnostic lab. This method is unique to pregnancy, due to the factthat EVT that are used here are the fundamental cells appearing onlyduring the window of pregnancy.

In another aspect, the invention includes a kit for assessing whether apregnant female is at risk of developing a disorder of pregnancyassociated with trophoblast invasion or placental ischemia. The kitincludes a natural or synthetic soluble matrix. Exemplary matricesinclude matrigel, collagen, fibronectin, elastin and combinationsthereof. Also included in the kit are labeled human endothelial cellsand labeled human trophoblast cells. The human endothelial cellsemployed may be from umbilical vein or they may be uterine, myometrial,cedicual, aorta, microvascular, dermal or other endothelial cells thatexpress VE-cadherin, PECAM, and/or Aquaporin 1. The trophoblastsemployed may be from primary villous or extravillous trophoblasts, HTR8,3A or JEG3 trophoblasts or other invasive trophoblasts isolated fromchoriocarcinomas. In using the kit, serum from the pregnant femalepatient is drawn. The endothelial cells and the trophoblast cells areco-cultured on the matrix and the appropriate amount (see above) of theserum is added. The culture is incubated for at least about 8 hours andthe results observed.

In yet another aspect, the assay of the invention is amenable tohigh-throughput screening, either for the identification of compounds orsubstances (i.e., agents) useful in the treatment of the aforementionedpregnancy disorders or conditions or useful as contraceptives orabortificients. This can be achieved by modification of publishedmethods, for example, see Withington.⁽³³⁾ Briefly, labeled endothelialcells and labeled trophoblasts are plated on an appropriate matrix. Thecells are stimulated with preeclampsia serum (to identify compounds orsubstance to treat pregnancy disorders) in the presence or absence ofthe test compound or substance, and incubated for an appropriate periodof time, typically 12 to 14 hours, under appropriate incubationconditions. After incubation, the cells are observed and analyzed todetermine whether the test compound or substance has stimulated the duelcell cross-talk between the endothelial cells and the trophoblasts ascompared to the control. PES is expected to disrupt the cross-talkbetween endothelial cell and trophoblasts while potential therapeuticsthat can reverse this disruption will be beneficial in the treatment ofthe aforementioned disorders or conditions. Duel cell cross-talk can bequantified based on the ratio of the two florescence signals. Toidentify agents useful as contraceptives or abortificients, the samemethod described above may be employed with one modification: in thisscreen normal pregnancy serum is employed instead of preeclampsia serumfor stimulation. Normal pregnancy serum is expected to support thecross-talk between endothelial cells and trophoblasts while compounds orsubstances that disrupt this architectural imprinting will be useful ascontraceptives or abortificients.

In yet another aspect, the invention provides a humanized in vivo animalmodel using interleukin-10 (IL10) null animals that was found to behighly sensitive to predict clinical symptoms of preeclampsia likeproteinuria, elevated blood pressure and kidney pathology, using asingle injection of serum from pregnancy. The term “humanized” as usedherein refers to the mimicking of the onset of human conditions in theanimal using human material. In the present case, administration ofhuman serum samples induced preeclampsia like symptoms in pregnant IL1-0 null mice; however other rodents, including guinea pigs and hamstersmay be employed as long as they are deficient in IL 10 expression. Thismodel can be used for high throughput screening for the discovery ofmolecules that can restore the serum-induced disrupted cross-talk andalleviate preeclampsia and/or other disorders of placental ischemia.Such examples include but not limited to recombinant human proteins andamino acids like rH VEGF A, rH VEGF C, hCG, TGF Beta, low molecularweight heparin and other small synthetic, semi-synthetic or naturalmolecules. Administration of potential cross-talk restoring agents to ananimal that has previously been administered human PE serum and isexhibiting PE-associated symptoms, and observing whether thePE-associated symptoms are alleviated thereby provides a method forscreening for agents to treat PE and/or other disorders of placentalischemia.

In developing the assay of the invention, it was observed thatcomplement split C5a levels are elevated in pre-eclampsia two-fold abovethe normal values seen in pregnancy. Further, data showed that the onsetof preeclampsia appears to be related to the levels of C5a. We alsodiscovered, unexpectedly, that such elevated C5a levels are detrimentalto the proactive cross-talk between trophoblasts and endothelial cellsinhibiting the vacuolization, which in clinical settings can lead toreduced trophoblast invasion of spiral arteries (see FIG. 9). Incombination with elevated sFlt-1 and soluble endoglin, elevation of C5awas found to synergistically become functionally lethal totrophoblast-endothelial cross talk at the fetal-maternal interface (FIG.9). Thus, controlling increases in maternal C5a levels is a therapeuticmethod in the treatment of pregnancy disorders such as preeclampsia.Furthermore, modulators of maternal C5a levels are suitable compositionsfor therapeutic intervention for preeclampsia and other disorders ofpoor trophoblast invasion resulting in placental ischemia. Examples ofsuch modulators include peptides, N-acetylated macrocyclic peptide 3D53,cyclic peptide derivatives and other C5a antagonists⁽³⁴⁾.

Similarly, other proteins expressed on the trophoblasts or endothelialcells or both are required for normal cross-talk and architecturalimprinting of trophoblasts on endothelial cells. Such proteins areexemplified by aquaporins (eg. aquaporin-1), Delta like ligand (eg.DLL4), whose function, when blocked using specific monoclonalantibodies, was found to disrupt cross-talk between endothelial cells ortrophoblasts. Since preeclampsia is trophoblastic disease with shallowtrophoblast invasion, such proteins are targets amenable to therapeuticmanipulations so as to facilitate complete and optimal trophoblastinvasion leading to normal pregnancy. Molecules that can modulate thesetargets can be screened based on the described method and used to treatdisorders of placental ischemia resulting from poor trophoblast invasionsuch as preeclampsia, gestational diabetes, intrauterine growthrestriction and placental abruption.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C are photographic representations of the effect of normalpregnancy serum at 37-40 weeks on endothelial cells as described inExample 1. FIG. 1 a shows the endothelial cells as a monolayer on a twodimensional culture, FIG. 1 b shows endothelial cell architecture on athree dimensional basement matrix-matrigel and FIG. 1 c shows thekinetics for the architectural rearrangement for endothelial cells.

FIG. 2 is a photographic representation of the effect of normalpregnancy serum on trophoblast cells as described in Example 2.

FIG. 3 is a photographic representation showing that NPS supports atrimester-specific differential interaction between trophoblasts andendothelial cells as described in Example 3.

FIG. 4 is a photographic representation showing the results of Example 4demonstrating that preeclampsia serum disrupts the interaction oftrophoblasts and endothelial cells. Large vacuoles and substantiallysmaller branch points can clearly be seen

FIG. 5 is a photographic and a graphic representation of the results ofthe experiment described in Example 4 showing the quantitative effect ofNPS, mild PE and severe PE on first trimester trophoblast-endothelialcell co-cultures.

FIG. 6 is a graphic representation of the results of the cytotoxicityassay described in Example 5.

FIGS. 7A-C are photographic and graphic representations of the resultsof the longitudinal studies of endothelial-trophoblast co-culturesdescribed in Example 6.

FIG. 8A is a photographic representation of the results of endothelialcells and HTR8 trophoblasts co-cultured and stimulated with pregnancyserum with or without recombinant human complement split product C5a, asdescribed in Example 7.

FIG. 8B is a table showing complement split product C5a levels areelevated on serum samples from preeclampsia.

FIG. 9 is a photographic representation of the results of endothelialcells and HTR8 trophoblasts co-cultured and stimulated with normalpregnancy serum with or without sFlt-1, sEng, or C5a, as described inExample 7.

FIG. 10 is a photographic representation of the results of endothelialcells and HTR8 trophoblasts co-cultured and stimulated with normalpregnancy serum or severe PE serum with or without TGFβ as described inExample 8.

FIGS. 11A-B are a graphic representation and a photographicrepresentation of the results of the experiment described in Example 9in which PE serum induced disrupted vacuolization is reversed byaddition of certain hormones and growth factors.

FIG. 12 is a photographic representation of the results of theadministration of normal pregnancy serum and preeclampsia serum onpregnancy outcome (gestational day 17) in wild type and IL-10 null miceas described in Example 10.

FIG. 13 is a table showing the effect of normal pregnancy serum andpreeclampsia serum on blood pressure, proteinuria, fetal weight andnumber, systemic levels of sF1t-1 and sEng in IL10 animals.

FIG. 14 is a table showing the effect of normal pregnancy serum andpreeclampsia serum on blood pressure, proteinuria, fetal weight andnumber, systemic levels of sF1t-1 and sEng in symptoms in WT animals.

DETAILED DESCRIPTION

Mammalian reproduction involves a complex, highly choreographed set ofmolecular processes that include interactions between the hormonallystimulated uterus and the developing blastocyst, implantation, a periodof placental and fetal development, and a terminal pathway composed ofdecidual/membrane activation, myometrial contractility, and cervicalripening⁽¹⁻³⁾. While metabolic changes and the placentalmicroenvironment are programmed in a pregnancy compatible manner,pregnancy presents itself as an immunological and hormonal paradox⁽⁴⁾.The role of estrogen and progesterone is well known in uterinereceptivity, implantation, local immune modulation, and early pregnancysuccess⁽²⁾. Insulin resistance similar to that imparted by inflammatoryresponses in non-pregnant individuals is exhibited during pregnancy⁽⁵⁾,which leads to the sustained transplacental nutrient flux required forfetal growth and development. Trophoblast invasion of the decidua isconsidered central to the free flow of nutrients and blood to the fetus.A poor perfusion of placenta, defective trophoblast invasion andremodeling of spiral arteries, and ensuing placental ischemia have beenthought to be associated with intrauterine growth restriction (IUGR) andpreeclampsia (PE).

Initial evidence supported the hypothesis that alterations inendothelial function were responsible for the pathophysiology ofPE^((5, 10)). However, reassessment of the disorder has led to a newtheory involving the placenta and secondary maternal illness ofhypertension, proteinurea and edema (5). PE remains highly heterogeneousand dangerous late pregnancy complication. A key factor appears to bethat the disease is a pregnancy and placenta-specific syndrome. Althoughendothelial dysfunction is a major change, it appears to be aconsequence of the interaction between poor placental perfusion andmaternal factors resulting in systemic inflammatory syndrome^((5, 10)).As a matter fact, normal pregnancy is also associated with somewhatelevated inflammatory responses such as insulin resistance, leptinproduction and apoptotic or necrotic trophoblast debris incirculation⁽¹¹⁾. Thus, one hypothesis is that PE is an extrememanifestation of mild inflammatory conditions typical of normalpregnancy. These observations suggest that PE may also represent animbalance between inflammatory and anti-inflammatory milieu duringpregnancy.

To establish a link between preeclampsia, placenta-endothelialdysfunction, and systemic inflammatory syndrome, it is tempting tohypothesize that the factors that choreograph co-onset of local defectsat the maternal fetal interface and systemic manifestation ofPE-associated symptoms are present in patient's circulation. Thisimplies that serum from PE patients and normal pregnancy subjects couldprovide a “blueprint” of etiologic factors. The maternal factor shouldoriginate in the placenta, must be disseminated to circulation, and behighly elevated during gestation because it disappears after thedelivery of the placenta. Although in vitro studies suggest that serumfrom a sub group of PE patients causes apoptosis in trophoblast cellsdue to activation of complement cascade (our unpublished data), anappropriate animal model should allow for its in vivo evaluation and foridentification of the exact nature of the placenta derived maternalfactor(s) in vitro.

The choice of animal model for experimental studies of adverse pregnancyoutcome in response to environmental factors is limited because of highvariability in the reproduction biology of mammalian species⁽¹²⁾. Therequirement for a closely related animal is particularly importantbecause, where possible, identical phenotypic and molecular biomarkersshould be monitored in both human and animal models. These requirementsplus the availability of immunological, hormonal, and molecular reagentslimit the choice of a model of mice. The similarities between human andmouse pregnancy begins with the hormonal regulation of uterinereceptivity for blastocyst implantation⁽²⁾. This is followed byhemochorial placentation, recruitment of phenotypically and functionallysimilar immune cells, placental production of cytokines, chemokines andhormones, and spiral artery remodeling by invading trophoblasts. We haverecently used wild type and knockout strains of mice to study theimmunobiology of pregnancy in response to inflammatory agents andcytokine deficiency⁽¹³⁾. Such studies provide important information onbiological assays and molecular targets relevant to human studies.

Since local and systemic features of PE are inter-dependent, serum frompregnancy can provide a “blueprint” of PE pathology and mimic clinicalsymptoms associated with PE, specifically, elevated blood pressure,proteinuria, kidney pathology, and IUGR, in an appropriate animal model.A small number of experimental models have included mouse strains thatare either genetically modified in growth or angiotensinogen regulatorypathways, or administered with treated leukocytes, but do not addressthe issue of intrinsic defects or local/systemic inflammation⁽¹⁴⁻¹⁷⁾. inour model, a single in vivo administration of serum from PE patients isemployed in pregnant, wild-type mice without a predisposed condition.Importantly, this serum-based model is pregnancy specific, as PE serumdoes not cause any ill effects such as elevated blood pressure andproteinurea in non-pregnant animals

The observed vascular deficiency in PE reflects a defective cross-talkbetween invading trophoblasts and the spiral artery endotheliumresulting in poor spiral artery remodeling and free flow of nutrients tothe fetus. Because of co-onset of local placental anomalies and systemicmaternal condition, we hypothesize that serum from PE patients canprovide a “blueprint” of causative factors. We further propose that amouse model and an in vitro model of endothelial cell-directedfingerprinting of trophoblasts mimicking spiral artery remodeling can beestablished to unravel the intrinsic ability of serum to induce in vivoor in vitro hallmark PE-associated characteristics.

Normal pregnancy involves the close interaction between placental cellsand maternal cells led by migration of trophoblasts into the maternaltissue. Pregnancy specific complications like preeclampsia, ischaracterized by shallow trophoblast invasion of the maternal spiralarteries. Trophoblasts are pregnancy specific stem cells representingfetus, while the endothelial cells represents the functional cells ofmaternal spiral arteries. Serum represents a unique media that hostsnumerous bio-markers that mirrors the global welfare of fetus and motherin successful pregnancy. Thus, serum can “blueprint” any anomalies veryearly during the pregnancy, much before clinical diagnosis of the onsetof complications.

The following examples illustrate certain exemplary aspects andembodiments of the invention and are included for illustration purposes.

Example 1 Effect of Normal Pregnancy Serum (NPS, 37-40 Weeks) onEndothelial Cells

Human umbilical cord endothelial cells (HUVEC) and human uterineendothelial cells (HUtEC) were obtained from Cambrex (East Rutherford,N.J., USA). Growth factor-reduced Matrigel representing basementmembrane (BD Biosciences, San Diego, Calif.₁USA) was thawed overnight at4° C. and mixed to homogeneity. The 48-well culture plates (Costar) werecoated with 0.1 ml of Matrigel and allowed to gelatinize at 37° C. for30 min. 2.5×10⁴ endothelial cells (HUtEC or HUVEC) labeled with celltracker orange CMTMR (Molecular Probes, Eugene, Oreg.) were plated (1:1)in the presence of RPMI media containing 10% human pregnancy serum onthe matrigel-coated plates. The architectural and morphological changesthat took place were monitored and recorded 12-14 hrs after incubationunder standard culture conditions using florescence microscopy (4×magnifications, Nikon Eclipse TS 100 coupled with CCD camera;photographs take every two hours). The endothelial cells migrated,underwent cytoskeletal reorganization, and formed tube-like capillarystructures impregnated with vacuoles and branch points where they assumestructures similar to their in vivo morphology. See FIG. 1 c. Thenumbers of completely formed vacuole were recorded manually as number oftubes in four different fields of view and defined as vacuolization. Anaverage of 45+6 vacuoles/well of a 48 well plate are formed. FIG. 1 ashows the endothelial cells as a monolayer on a two dimensional cultureand FIG. 1 b shows endothelial cell architecture on a three dimensionalbasement matrix-matrigel. FIG. 1 a-c are copies of photographs taken att=12 hours.

Example 2 Effect of Normal Pregnancy Serum on Trophoblast Cells

The first trimester human trophoblast cell line HTR8 (representingnormal invasive extravillous trophoblasts), the third trimester humantrophoblast cell line TCM, and the first trimester human villoustrophoblast cell line 3A were used in the following studies. Human HTR-8cells and TCI-1 cells were a gift from Dr. Charles Graham (QueensUniversity, Canada) and human 3A cells r were a gift from Dr. Gil Mor,Yale University, USA.

Cytotrophoblasts from human placental tissue were isolated according topublished methods. Briefly, placental tissues were digested withdecreasing concentrations of trypsin-DNase 1 (trypsin, 1 mg/ml; andDNase, 1.5 mg/ml) at least four times at 37° C. for 20 min each. Thecells from the first digestion were excluded. The cell mass collected inthe following steps was treated with a lysis buffer (0.15 M NH₄Cl, 1 mMKHCO₃, and 0.1 mM EDTA (pH 7.3) for 5 min at room temperature withconstant shaking to lyse the red blood cells (RBCs), which if notremoved disturb separation on Percoll gradients. Cytotrophoblastsisolated in this manner were stained for cytokeratins or CD45 (a markerfor immunocytes) to ascertain their purity (>95%).

Both freshly isolated primary trophoblasts (isolated as described aboveand cultured overnight) and trophoblast cell lines HTR8, TCI-1 and 3Awere maintained in Roswell Park Memorial Institute (RPMI) 1640 mediapurchased from Gibco (Gaithersburg, Md., USA) supplemented with 10%fetal bovine serum, 2 mM L-glutamine (Gibco BRL), 100 U/ml penicillin(Gibco BRL) and 0.1 mg/ml streptomycin (Gibco BRL). Cell lines weregrown to ˜80% confluence and less than eight passages were used in thestudy. HUVEC and HUtEC cells were maintained in EBM-2 and used withinsix passages. All cells were maintained in standard culture conditionsof 5% CO₂ at 37° C. 2.5×10⁴ trophoblast cells in different trimesters,labeled with cell tracker green CMFDA (Molecular Probes, Eugene, Oreg.),were plated on Matrigel-coated plates and stimulated with mediacontaining 10% NPS as described in Example 1. The architectural andmorphological changes that took place were recorded 12-14 hrs afterincubation under standard culture conditions using florescencemicroscopy (4× magnifications, Nikon Eclipse TS 100 coupled with CCDcamera; photographs take every two hours). None of the trophoblast cellsformed capillary tube like structures on matrigel in response tostimulation with NPS, reflecting their inherent behavior in vivo. Theseresults are shown in FIG. 2, which is a reproduction of a photographtaken at the end of the experiment.

Example 3 NPS Supports Trimester-Specific Differential Interaction withEndothelial Cells

2.5×10⁴ endothelial cells labeled red and trophoblasts from humansubjects, each from different trimesters labeled green, were co-culturedon matrigel-coated plates and stimulated with Normal Pregnancy Serum(NPS) (sample L31, see Table 1) as described in Examples 1 and 2.Gestational age specific architectural patterns were observed. As seenin FIG. 3, left panel (which is labeled “EC+HTR8+L31”), surprisinglyonly the first trimester HTR8 trophoblasts spontaneously interact andalign with endothelial cell architecture. S3A cells, representingvillous trophoblasts from first trimester, attract endothelial cells andform massive branch points that allow reduced vacuolization only aroundits vicinity. (See FIG. 3, middle panel, labeled “EC+3A+L31.”) Incontrast, term trophoblasts and third trimester TCL-1 trophoblasts tendto naturally impede the capillary formation by endothelial cells.Smaller vacuoles result. (See FIG. 3, right panel labeled“EC+TCL-1+L31.”) Thus, NPS supports the proactive cross-architecturalrealignment of endothelial and first trimester trophoblast cells, butdoes not support realignment of the cells in the third trimester and atterm.

Example 4 Preeclampsia Serum Disrupts Gestational Age-SpecificDifferential Interaction with Endothelial Cells

Blood samples were obtained from normal pregnant human subjects andpreeclamptic human subjects during first (6-12 weeks), second (13-20weeks) or third (21-40 weeks) trimester of pregnancy and serum separatedroutinely. Pregnancies were considered normal when there were no medicalcomplications. Preeclampsia was defined when blood pressure was >140/90mm Hg at least on two occasion 4 hours to 1 week apart and withproteinuria >300 milligram in 24 hr urine collection. Exclusion criteriawere chronic hypertension, diabetes, antiphospholipid antibody syndrome,thrombophillic anomalies, antepartum and postpartum complications. Allthe studies were approved by the human institutional review board at theWomen and Infants Hospital of Rhode Island. The samples were analyzedfor content of sFlt-1, sEng, C5a and hCG levels using commerciallyavailable respective ELISA kits according to manufacturers protocol. Theprofiles of representative samples are provided in Table 1.

TABLE 1 Profiles of representative human pregnancy serum samplesindicating gestational week of collection, onset of disease and severityof disease. Preeclampsia/ Gestational Number Normal Week Onset SeverityL32 Normal L31 Normal L45 Normal L1045 Normal 39 L1037 Normal 35 L1051Normal 40 L12 Preeclampsia 35 L1015 Preeclampsia 38 late severe L1035Preeclampsia 42 late moderate L1048 Preeclampsia 33 early severe L1Preeclampsia 34 early severe L1020 Preeclampsia 36 late severe L1027Preeclampsia 40 late moderate L35 Normal L1024 Preeclampsia 41 latemoderate P118 Normal PS318 Preeclampsia 7 severe PS299 Preeclampsia 27mild PS303 Preeclampsia 14 severe PS334 Preeclampsia 26 mild Pre13Preeclampsia 36 mild

2.5×10⁴ endothelial cells labeled red and 2.5×10⁴ trophoblasts, eachfrom different trimesters labeled green, were co-cultured on matrigelcoated plates and stimulated with either NPS or mild or severe PE serum(PES refers to Preeclampsia Serum) as described in previous examples forNPS alone. Exemplary results are shown in FIG. 5. Serum from either mild(sample L1035; middle left panel) or severe (sample L31; bottom leftpanel) preeclampsia patients blocks the “cross-talk” betweenendothelial-trophoblast cells, causing obvious differences inarchitecture as compared to the same cells stimulated under the sameconditions with NPS serum (sample L31; top left panel). Preeclampsia canbe classified as mild or severe. Severe preeclampsia is characterized by(1) a systolic blood pressure greater than 160 mm Hg or diastolic bloodpressure greater than 110 mm Hg on 2 occasions at least 6 hours apart ina woman on bed rest and (2) the presence of significant proteinuria.Marked proteinuria is defined as 5 g or more of protein in a 24-hoururine collection. Severe preeclampsia, at times, may be associated witholiguria, cerebral or visual disturbances, pulmonary edema or cyanosis,epigastric or right upper quadrant abdominal pain, impaired liverfunction, and thrombocytopenia. In mild preeclampsia (or moderate PE),hypertension and proteinuria are present, but not to these extremelevels, and the patient has no evidence of other organ dysfunction, (seehttp://www.emedicine.com/med/topic1905.htm Preeclampsia (Toxemia ofPregnancy)).

The number of vacuoles (or tubes) formed per sample were counted.Exemplary results are shown in the graphic panel on the right. Ascompared to the control serum free media (SFM) and NPS, the cellsco-cultured with either mild or severe PE serum exhibited significantdecreases in capillary tubes formation (63 versus 35 and 26).Statistical significance of experimental differences was assessed usingStudent's paired t-test. The differences were considered to bestatistically significant when the p value was <0.05.

Example 5 Cytotoxicity Assay

Since disruption of tube/vacuole formation could have been due to celldeath induced by toxic components present in the human serum, acytotoxicity assay was performed as follows. Growth factor-reducedMatrigel (representing basement membrane) was obtained (BD Biosciences,San Diego, Calif., USA), thawed overnight at 4° C., and mixed tohomogeneity. 48-well culture plates (Costar) were coated with 0.1 mlMatrigel and allowed to gelatinize at 37° C. for 30 min. HTR8trophoblasts or endothelial cells (2.5×10⁴) were plated (1:1) in thepresence of serum free media containing 10% human normal pregnancy serum(NPS) severe preeclampsia serum (PES) or mild preeclampsia serum (mPE).After overnight incubation, the cells were isolated from the Matrigelusing BD cell recovery solution (BD Biosciences, USA), stained withpropidium iodide (PI) solution (0.1%), and analyzed by flow cytometry.Live cells do not take up PI while dead cells are stained by red colorflorescence that can be quantified and expressed as % PI positive (dead)cells by FACS (BD Canto, BD Biosciences, USA). The results expressed aspercentage of dead cells as indicated by the number of cells that havetaken up propidium iodide stain (PI) out of total cell population andare illustrated in FIG. 6. Both the normal pregnancy serum (NPS) and theserum from mild and severe preeclamptic human female patients exhibitedsubstantially low percentages of dead cells (about 15%) than normal, andis comparable to RPMI media without the presence of serum (Serum-freeMedia SFM). Neither the endothelial cells nor HTR8 cells were killed bythe preeclampsia serum samples. Thus, the disruption of thecharacteristic architecture formed by the endothelial cells and HTR8trophoblasts is not due to cell death.

Example 6 Quantitative Effect of NPS, Mild PE and Severe PE on FirstTrimester Trophoblast-Endothelial Cell Co-Cultures

Longitudinal studies with PE serum on endothelial-trophoblastco-cultures were next undertaken. 2.5×10⁴ endothelial cells and 2.5×10⁴HTR8 trophoblast cells were co-cultured on matrigel, stimulated withhuman PE serum collected at different weeks of pregnancy and incubated12-14 hours. The number of vacuoles/tubes formed and signature patternof architectural morphology were recorded. FIGS. 7 a and 7 b representthe signature architectural morphology of the co-cultures whosephotographs were taken at the end of the incubation (12-14 hrs) inresponse to serum samples collected at different stages of pregnancy(see Table 1 for the descriptions of type of serum (mild or severe PE orNPS) used). Left and right panels represent photographs (duplicate)taken from two different field of view of a well. The number of vacuoles(or tubes) formed per sample were counted and the results are shown inFIG. 7 c in which, the week of blood draw and pregnancy outcome is alsoindicated below the boxed graphic. The results illustrated in thesefigures demonstrate that preeclampsia serum disrupts the characteristic“architectural imprinting” of HTR8 and endothelial cells as early as7-14 weeks of pregnancy and can therefore be used as an early predictivetool to assess likelihood of a patient developing preeclampsia.Statistical significance of experimental differences was assessed usingStudent's paired t-test. The differences were considered to bestatistically significant when the p value was <0.05.

Example 7 Serum Factors from PE that Contribute to Abnormal Endo-TrophoCross-Talk: Synergistic Effects of Complement Split Product C5a withSoluble Factors

Previous studies (reference 18 and cross references therein) indicatethat PE is associated with elevated levels of the soluble form of Flt-1(sFlt-1), also known as soluble vascular endothelial growth factor R1(VEGF R1). sFlt-1 is an alternate splice variant of VEGF R1, whichexhibits high affinity for growth factors like VEGF and placental growthfactor (PlGF) and binds to these proteins, blocking their functionalactivities. Similarly, endoglin (also known as CD105) is a co-receptorfor transforming growth factor beta (TGFβ). Soluble forms of endoglin(sEng) bind TGFβ and impair its signaling. Surprisingly, exogenousspiking of sFlt-1 or sEng at levels present in normal pregnancy serum orin preeclampsia serum fails to disrupt the cross-talk betweentrophoblasts and endothelial cells. For this reason, we expected that PEserum would affect the vacuolization of endothelia-trophoblastsirrespective of sFlt-1 or endoglin levels. Given the possibility thatthese soluble factors are not the cause but consequence of an upstreamfactor, we next analyzed levels of sFlt-1, sEng, and complement splitproduct C5a in the normal, severe, and mild pregnancy serum samples setforth in Table 1 (see Example 4). As shown in Table 2, severe PE serumshows a statistically significant elevation of C5a, sFlt-1, and sEnglevels that reflects at both early and late onset of the disease.

TABLE 2 Complement split products C5a are elevated in serum samples frompreeclampsia. sEng(ng/ml) sFlt-1(ng/ml) C5a(ng/ml) Normal (n = 25) 16.93 ± 18.17  14.4 + 4.33  9.77 + 29.19 Mixed PE 145.88 ± 41.38** 1.43 + 11.6** 53.44 + 37.73^(a) Population (n = 37) Severe  56.68 ±38.26** 26.89 + 11.92*  7.94 + 30.14^(b) (n = 11) Mild (n = 26)  I41.38± 42.57^(a) 19.15 + 10.91**  1.57 + 40.92 Early Onset  67.56 ± 45.00^(a)25.57 + 14.76** 48.91 + 22.53^(c) (n = 9) Late Onset   39.21 + 37.94^(a)20.15 + 10.26** 54.84 + 40.77^(a) (n = 28) All values are expressed asmean +/− sd. **, a, b, c indicates statistical significance at P <0.001, P < 0.01, P < 0.02, P < 0.07. Parenthesis indicates the number ofserum samples analyzed.

As indicated in Table 2, the values of sFlt-1, sEng, and C5a arestatistically much higher in preeclampsia serum compared to normalpregnancy serum, irrespective of disease phenotype. Importantly, sFlt-1and sEng levels are substantially lower in mild as compared to severepreeclampsia. We sought to explore the effect of C5a onendothelial-trophoblast cross-talk experimentally as described below.

Briefly, 2.5×10⁴ labeled endothelial cells and 2.5×10⁴ HTR8 trophoblastswere co-cultured on matrigel coated plates, stimulated with normal humanpregnancy serum with or without 100 ng/ml recombinant human complementsplit product C5a, and incubated for 12-14 hours as described inprevious examples. The signature patterns of architectural morphologywere recorded. Exemplary results are shown in FIGS. 8A-B for sample L35.NPS spiked C5a disrupts the endothelial-trophoblast cross-talk (panel“B”), indicating that imbalance in complement cascade split productscould be one of the causes for the loss of activity. Further, to assessthe complementary role of C5a to the presence of sFlt-1 and sEng, thefollowing experiment was carried out.

Labeled endothelial cells and HTR8 trophoblasts (2.5×10⁴ each) wereco-cultured on matrigel and stimulated with normal pregnancy serum withor without sFlt-1, sEng, or C5a as described earlier. Exemplary resultsare shown in FIG. 9, which shown that NPS spiked with sFlt-1, sEng andC5a, in combinations, at physiologically relevant amounts can cause moredramatic and synergistic disruption of “endo-tropho cross-talk” andvacuolization of endothelial/HTR8 cell co-cultures than each of themindependently. This also demonstrates that the “cross-talk” betweenendothelial cells and trophoblasts cannot be disrupted by spiking NPSwith exogenous soluble Flt-1 or soluble Eng at concentrations seen ineither severe or mild pre-eclamptic serum unless there is elevated C5alevels that acts as catalyst to disrupt the cross talk.

Thus, these results provide direct evidence that C5a is possible targetamenable to pharmacological manipulations by which discovery oftherapeutic interventions can be sought.

Example 8 Low Dose TGFβ Rescues Preeclampsia Serum Induced Disruption ofCapillary Formation

2.5×10⁴ endothelial cells and 2.5×10⁴ HTR8 trophoblasts were co-cultured12-14 hours on matrigel and stimulated with normal pregnancy serum (NPS)or severe PE serum (PES) with or without TGFβ. Exemplary results forsamples L35 and L1 are shown in FIG. 10. As the pictorialrepresentations labeled A-D indicate, NPS support the vacuolization oftrophoblasts and endothelial cells and PES disrupts this cross talk. Ourdata also suggest that low doses of TGFβ, could rescue PES-disruptedtube formation, suggesting inhibition of sEng activity.

Example 9 Rescue of PE Serum-Induced Disruption of Cross-Talk by GrowthFactors and Hormones

2.5×10⁴ labeled endothelial and 2.5×10⁴ HTR8 trophoblast cells wereco-cultured on matrigel 12-14 hours and stimulated with either severe ormild PE serum, with or without 10 U/ml Heparin, 100 ng/ml VascularEndothelial Growth Factor A (VEGF A), 100 ng/ml Vascular EndothelialGrowth Factor C (VEGF C) or 10 mg/ml human chorionic gonadotropin (hCG).After 12-14 hours, the signature pattern of architectural morphology andnumber of tubes/vacuoles were recorded. The results are shown in FIGS.11A-B in which the graphic representation in FIG. 11 a indicates theaverage number of tubes formed by the co-cultured, severe PE stimulated(L1015, L1020 refer to Table 1), mild PE stimulated (L1024, L1027) cellscompared to the average number of tubes formed by the co-cultured, NPSstimulated (L32), cells. As seen in FIGS. 11A-B, NPS support thevacuolization while severe and mild PES disrupt the cross-talk.Co-incubation of mild PE serum with heparin can rescue the disruptedtube formation. Complement activity is generally inactivated by heattreatment. As indicated in FIGS. 11A-B, heat inactivated mild and severePE serum samples supported tube formation, supporting the notion thatcomplement factors are potential targets malleable to therapeuticintervention. Furthermore, these results indicate that exogenousaddition of pro-angiogenic recombinant proteins like VEGF A and VEGF Ccan indeed rescue inhibition of tube formation by PES, implying thatshallow trophoblast invasion and poor angiogenesis observed inpreeclampsia are due to lack of such factors in PES. Furthermore, hCGthough elevated in PE, does not seem to be functional. Using recombinanthCG, we show in FIG. 11 b, PES-induced disruption can be rescued byexogenous hCG. In FIG. 11 b, copies of exemplary photographs of theco-cultures are shown.

Example 10 Humanized Mouse Model for Predicting Hypertensive Disorders

The anti-inflammatory cytokine IL-10 plays a critical role in pregnancybecause of its regulatory relationship with other intrauterinemodulators and its wide range of immunosuppressive activities.Significantly, its local production by gestational tissues is welldocumented. We have demonstrated that IL-10 expression by the humanplacenta was gestational age-dependent, with significant expressionthrough the second trimester followed by attenuation at term. IL-10expression was also found to be poor in decidual and placental tissuesfrom unexplained spontaneous abortion cases, and from deliveriesassociated with preterm labor and preeclampsia (our unpublishedobservations). However, the mechanism(s) by which IL-10 protects thefetus remains poorly understood; IL-10^(−/−) mice suffer no pregnancydefects unless challenged with inflammatory agents. Since PES is able todisrupt trophoblast and endothelial cell functions, we hypothesize thatIL-10^(−/−) mice could provide a model system to establish a “humanized”model to study onset of preeclampsia like systems in mice.

Pregnant wild type or IL-10^(−/−) mice (C57BL/6, Jackson Labs, USA) wereinjected intraperitoneally on gestational day (gd) 10 with human normalpregnancy serum (NPS) or mild or severe PE serum (PES). On gd 16/17,urine and serum were collected from each mouse and blood pressuremeasurements were taken. Total urinary albumin was measured usingAlbumin (mouse) ELISA kit (ALPCO Diagnostics, Salem, N.H.) and urinarycreatinine was measured using Metra Creatinine Kit (Quidel Corporation,San Diego, Calif.). Proteinuria is represented as the ratio of urinaryalbumin to creatinine (expressed as μg/mg). The baseline values seen inmice ranges from 100-400 μg/mg. Blood pressure was examined by anestablished tail-cuff method which utilizes a programmedsphygmomanometer. The animals adapted for 5 min using a warming testchamber (MTC Life Science Inc, Woodland Hills, Calif.) at controlledtemperature (35° C.). The measurements were carried out on day 17 ofpregnancy using DigiMed blood pressure analyzer, (MicroMed, 8008 VineCrest Avenue, Suite 3, Louisville, Ky., 40222-4683). Each measurement ofblood pressure is an average of three readings at 1 min interval from anumber of animals (˜3-5 each). Systolic blood pressure was comparedamong non pregnant and pregnant mice. All animals were age matched. Datawas analyzed using Digi-Med® System IntegratorTM Model 400 (DMSI-400).The data are provided in Tables 3 & 4.

TABLE 3 Effect of intraperitoneal injection of normal pregnancy serumand PE serum on BP, number of fetus and fetal weight, urine levels ofproteinuria and serum levels of sFlt-1 and sEng levels in IL10 knockoutmice. sFlt-1 sEndoglin Systolic BP Proteinurea Aev Fetal (ng/ml) (ng/ml)(mmHg) (ug/mg) Wt (g) # Fetus NPS (n = 9) 61.25 ± 21.69 191.41 ± 45.3193.46 ± 3.3  145.68 ± 58.96 1.24 ± 0.04 9 ± 0.81 Severe PE 104.2 ± 28.7*395.31 ± 33.1*  128.0 ± 12.9*  391.89 ± 121.39*  0.91 ± 0.12* 8 ± 2  Serum (n = 4) Mild PE 55.29 ± 6.10  210.2 ± 24.2 113.48 ± 6.61* 264.01 ±94.8* 1.23 ± 0.09 9 Serum (n = 4)

TABLE 4 Effect of intraperitoneal injection of normal pregnancy serumand PE serum on BP, number of fetus and fetal weight, urine levels ofproteinuria and serum levels of sFlt-1 and sEng levels in wild typemice. sEndoglin Systolic BP Proteinurea Aev Fetal sFlt-1 (ng/ml) (ng/ml)(mmHg) (ug/mg) Wt (g) # Fetus NPS (n = 8) 68.8 + 21.6 176.5 + 148.4 102.7 + 28.1  387.28 + 162.9 1.27 + 0.09 8.33 + 1.5  Severe PE  51.9 ·28.18 379.4 + 172.3* 147.4 + 18.9*  828.5 + 570.9*  1.0 + 0.25  7.6 +1.51 Serum (n = 4) Mild PE 75.57 + 14.3  200.2 ± 21.2  101.9 + 17.58228.4 + 24.2 1.01 + 0.1  8 + 0 Serum (n = 4)

The results of the study summarized in Tables 3 & 4 suggest that anumber of PE serum samples representing mild and severe phenotypesinduced some or all PE-associated symptoms when injected i.p. ongestational day (gd) 10 in IL10 null C57 BL/6 ((IL10^(−/−)) and wildtype mice. The effects in response to only one administration of serum(100 μl) were evaluated on gd 17. Signature PE symptoms includingintrauterine growth restriction (IUGR) as reflected by reduced fetalweight, elevated systolic blood pressure (BP), proteinuria, and elevatedsFlt-1 and sEng levels were observed in response to severe PE serum andmild PE serum when compared to normal pregnancy serum (NPS).Significantly, severe PE serum samples had no effect on blood pressureand proteinurea in non-pregnant mice (data not shown). The highsensitivity and specificity of IL10 knockout mice to predictpreeclampsia is reinforced by the fact that mild preeclampsia serum caninduce hallmark symptoms of elevated blood pressure and proteinuria onlyin these mice but not in wild type animals. Statistical significance wasassessed using Student's paired t-test. Differences were considered tobe statistically significant when the p value was <0.05.

On gd 17 the mice were euthanized, the uterine horns were extracted andphotographed and pregnancy outcomes were recorded. Serum sFlt-1, andsEng were measured by ELISA using commercially available kits. Exemplaryresults are shown in FIG. 12 in which the upper left and right panelsare photographic reproductions of the uterine horns from IL-10 null andwild-type mice administered NPS, and the lower left and right panels arephotographic reproductions of the uterine horns from IL-10 null andwild-type mice administered severe PE serum [(100 μl). Black arrowsindicate sites of fetal resorption. As seen in the photographs, PE seruminduced intrauterine growth restriction (IUGR in FIG. 12) and fetalresorption in IL-10 knockout mice, and not in wild-type mice.

All patents, publications, and other references cited herein are herebyincorporated by reference. Although the invention has been particularlydescribed with reference to certain preferred embodiments, skilledartisans appreciate that changes in form and details may be made withoutdeparting from the scope of the appended claims.

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1. An assay for assessing whether a pregnant female is at risk of developing a pregnancy disorder characterized by either poor trophoblast invasion or placental ischemia, or both, comprising (a) incubating a co-culture of human endothelial cells and human trophoblast cells in the presence of serum obtained from a pregnant female for a period of time sufficient to permit vacuolization, and (b) after incubation, determining whether substantial vacuolization in the co-culture has occurred.
 2. The assay according to claim 1 wherein the disorder is selected from preeclampsia, gestational diabetes, intrauterine growth restriction, and trophoblasitic diseases with hyper- or hypo invasive features.
 3. The assay according to claim 2, additionally comprising the steps of incubating a co-culture of human endothelial cells and human trophoblast cells under the same conditions as the co-culture in claim 1(a) in the absence of serum or plasma from a pregnant female for a period of time sufficient to permit vacuolization and after incubation determining whether substantial vacuolization in either co-culture has occurred.
 4. The assay according to claims 1-3 wherein incubation time is at least 12 hours.
 5. The assay according to claim 4 wherein the serum is from the first trimester of the pregnant female's pregnancy.
 6. The assay according to claim 5 wherein the amount of serum used is 0.05-2 ml.
 7. The assay according to claim 6 wherein the endothelial cells are selected from umbilical vein, uterine, myometrial, decidual, aorta, microvascular, dermal or other endothelial cells that express VE-cadherin, PECAM, and/or Aquaporin
 1. 8. The assay according to claim 6 wherein the trophoblasts are selected from primary villous or extravillous trophoblasts, HTR8 and 3A trophoblasts or other invasive trophoblasts isolated from choriocarcinomas.
 9. The assay according to claim 7-8 wherein the co-culture is performed on a natural or synthetic soluble matrix.
 10. The assay according to claim 9 wherein the matrix is selected from matrigel, collagen, fibronectin, elastin and combinations thereof.
 11. The assay according to claim 10 wherein the incubation step is performed at 37° C. in carbon dioxide.
 12. A method of screening for agents to treat a pregnancy disorder characterized by either poor trophoblast invasion or placental ischemia, or both, comprising (a) administering a potential endothelial-trophoblast cross-talk restoring agent to an IL 10 null animal that has previously been administered human PE serum and is exhibiting PE-associated symptoms, and (b) observing whether the PE-associated symptoms are alleviated thereby.
 13. The method according to claim 12 wherein the animal is a mouse.
 14. The method according to claim 12-13 wherein the disorder of preeclampsia.
 15. A method of treating a pregnant female human patient suffering from a placental ischemia disorder comprising administering to the patient a therapeutically effective amount of a compliment split C5a modulating agent.
 16. A method of screening for agents useful in the treatment pregnancy disorders characterized by either poor trophoblast invasion or placental ischemia, or both, comprising (a) treating co-cultured endothelial cells and trophoblast cells with a potential endothelial-trophoblast cross-talk restoring agent in the presence of preeclampsia serum and in the absence of preeclampsia serum, and (b) comparing the results to determine whether the preeclampsia serum induced disruption of endothelial-trophoblast cross-talk is reversed thereby.
 17. A method of screening for agents useful as contraceptives or abortificients, comprising (a) treating co-culture of endothelial cells and trophoblast cells with a potential contraceptive or abortificient agent in the presence of normal pregnancy serum and in the absence of normal pregnancy serum, and (b) comparing the results to determine whether the normal endothelial-trophoblast cross-talk is disrupted thereby. 